This project will explore the regulatory system that governs progression through the stages of the eukaryotic cell division cycle, with an emphasis on the control of chromosome segregation in mitosis. The final events of mitosis are triggered by a poorly understood enzyme called the Anaphase-Promoting Complex or APC. The APC is a large, multisubunit ubiquitin ligase that catalyzes the ubiquitination of several key regulators of late mitotic events. In the proposed studies, biochemical and molecular genetic approaches will be used in the budding yeast Saccharomyces cerevisiae to dissect the enzymological mechanisms by which the APC, together with its various accessory components, recognizes its substrates and assembles polyubiquitin chains that direct those substrates to the proteasome for destruction. Three specific aims are proposed. The first aim focuses on the important question of how the APC recognizes its targets: mutagenesis and detailed biochemical analyses will be used to unveil the functions of three related APC subunits that contain a conserved sequence motif, the tetratricopeptide repeat or TPR, that forms a binding interface involved in substrate recruitment. The second aim centers on accessory enzymes that collaborate with the APC to help govern its function: recent studies from this laboratory have revealed that APC function depends on two ubiquitin-conjugating enzymes or E2s, and the proposed studies will explore the biochemical behavior of these two proteins. Additional work will address the function of a polyubiquitin chain-extending enzyme that was identified in preliminary studies as a potential collaborator of the APC. Finally, in the third aim, genetic and biochemical approaches will be used to understand how the ubiquitination state of APC targets is regulated by deubiquitinating enzymes in the cell. The knowledge gained from these studies will provide new insights into the control of chromosome segregation, errors in which often contribute to developmental problems and cancer progression. These studies are also likely to illuminate general mechanisms of protein ubiquitination, a regulatory modification of importance throughout cell biology and human disease.